apparent presence of ser133-phosphorylated cyclic amp res · apparent presence of...

1
Apparent presence of Ser133-phosphorylated cyclic AMP response element binding protein (pCREB) in brain mitochondria is due to cross-reactivity of pCREB antibodies with pyruvate dehydrogenase Jan Platenik , Vladimir J. Balcar , Yukio Yoneda , Barbara Mioduszewska , Richard Buchal , Radovan Hynek , Lukasz Kilianek , Nobuyuki Kuramoto , Grzegorz Wilczynski , Kiyokazu Ogita , Yoichi Nakamura , and Leszek Kaczmarek 1,2,3 2 2 3 1 4 5 2 3 6 2 3 99.12 1 2 3 4 5 6 Inst.Med.Biochem., 1st Fac. Med., Charles Univ., Praha 2, CZECH REPUBLIC. Lab. Mol. Pharmacol., Div. Pharm. Sci., Kanazawa Univ., Kanazawa, JAPAN. Dept. Mol. Cell. Neurobiol., Nencki Inst., Warsaw, POLAND. Dept. Biochem.&Microbiol., Inst. Chem. Technol., Praha, CZECH REPUBLIC. Lab. Confocal Microscopy, Nencki Inst., Warsaw, POLAND. Dept. Pharmacol., Setsunan Univ., Osaka, JAPAN. The cyclic AMP response element binding protein (CREB) is an ubiquitously and constitutively expressed transcription factor of the basic/leucine zipper family that recognizes a DNA consensus sequence called CRE (cyclic AMP/Ca2+ response element). Its ability to activate transcription of the regulated gene critically depends on phosphorylation of a serine residue number 133 in its transactivation domain, which is done by variety of protein kinases in response to elevation of intracellular calcium, cAMP or neurotrophin signaling. With over 5000 reports related to CREB in Medline to date this protein has also become one of the most studied transcription factors. In neuroscience the CREB has been widely implicated in the synaptic plasticity underlying long-term memory consolidation, and also in the neuronal pro-survival signaling pathways. The prevailing view is that CREB is permanently and exclusively present in the cell nucleus. However, there have been several mutually controversial reports describing an extranuclear localization of CREB. Specifically, synaptic, cytosolic, and in particular mitochondrial localization was reported. Here we present results of our own study on subcellular localization of CREB. Introduction Methods The subcellular localization of CREB was examined by means of: - Subcellular fractionation of adult rat brain cortex followed by Western imunoblotting with CREB/pCREB antibodies and gel shift with CRE probe. Suitable nuclear, mitochondrial and synaptic markers also employed. - Immunofluorescence staining and confocal microscopy of cultured rat brain neuronal and astroglial cells - Immunoprecipitation of CREB or pCREB IR from nuclear or mitochondrial fractions, followed by Western blotting analysis and identification of precipitated proteins by MALDI-TOF mass spectrometry The following commercial CREB/pCREB a ntibodies (Ab's) were used: Pho spho- CREB (S er13 3) Ab, Ce ll Signalling Techno logy (CS T, #9 191) Phospho-CREB Ab from Upstate Biotechnology (#06-519) CREB antibody (against an epito pe at the CRE B C-terminus), CST(#9192) C-21 CREB Ab (against CREB DNA-binding domain), Santa Cruz Biot echnology (sc-186). HmCREB antibody (from "home-made") was raised in Y. Yoneda's labo ratory against pep tide KRREILSRRP SYRKC (residues 123-136 of mouse or rat CREB, encomp assing u nphosphorylated Ser133), and affinity-purified using the sam e peptide. (pCREB... CREB phosphorylated at Ser133, IR... immunorea ctivit y, MALDI-TOF... m atrix-assisted laser desorptio n/ionisat ion ti me-of-flig ht) 1) Subcellular fractionation procedure Fractions obtained: H P1 P2 S P3 A B C D E (original homogenate) (nuclear fraction, but also containing a lot of mitochondria from cell debris) (crude mitochondria/synaptosomes) (cytosol) (microsomal) (probably cell membranes, Golgi) (synaptosomes) (non-synaptic mitochondria) (synaptic membranes, but also containing a lot of synaptic mitochondria) (synaptic mitochondria) Protein concentration and yield Western blotting for CREB, pCREB, NMDA receptor 2B subunit Cytochrome oxidase activity Gel shift for CRE DNA binding activity In each fraction measured: 2) pCREB IR is prominent in mitochondrial fractions, but CREB IR is not A), C), D): representative Western blots of the subcellular fractions probed with various pCREB and CREB antibodie s as indicated. B): Densitometric evaluation of the pCREB Ab C ST IR (inset) in comparison to distribution of NMDA recep tor 2B subunit IR (synaptic marker) and azide-inhibitable cytochrome oxidase enzyme activity (mitochondrial marker). Me an ± SD, N=4 independent experimen ts, only for cytochrome oxidase N=2 independent experiments. *…p<0.05, **… p<0.01 vs. original homogenate), one-w ay ANOVA with Dunnett's pos t test. The signal with two different pCREB Ab's was strong in mitochondria-enriched fractions (C, D, E in particular), and quantitative distribution of pCREB IR correlated with the mitochondrial marker rather than synaptic one. In contrast, the band detected with two CREB antibodies against different epitopes was seen mostly in nuclear fraction, some in S and P3, but almost absent from mitochondrial fractions. 3) Gel shift: mitochondrial/synaptic CRE DNA binding activity different from nuclear CREB Gel shift with 32P-labelled CRE probe performed with all the subcellular fractions, for nuclear and mitochondrial fractions also ncluding supershift with various CREB/pCREB Ab's as indicated). The dominant lower band appearing in the nuclear fraction was supershifted by two CREB Ab's which qualifies this band as authentic CREB. In contrast, the CRE- binding activity in mitochondrial/ synaptic fractions differed in electrophoretic mobility and was unaffected by CREB Ab's. 4) Nuclear contamination of P2 fraction was low but detectable DAPI stain of freshly prepared P1 and P2 fraction revealed a plenty of nuclei and blood vessel fragments in the P1, but only very few nuclei in the P2. Likewise, presence of histone H3 detected with Western immunoblotting was restricted to the P1 f raction. Several dilutions of P1 fraction were included to show that the faint histone band in P2 actually did not represent more than about 1% of histone conce ntration in P1. 5) Electrophoretic mobility of mitochondrial pCREB IR markedly differed from nuclear pCREB/CREB IR A): The CREB IR on Western blots de tected with the CREB Ab CST in the nuclear fraction consistently appeared as a double band, likely representing two CREB splicing variants, alpha (the upper band) and delta (the lower band). All other fractions apparently contained only delta CREB. The blots detected with pCREB Ab Upstate also displayed a non-constant double band in the nuclear P1 fraction, while in the mitochondrial P2 fraction there was always a single band with position corresponding to the P1 lower band. B) 2-D electrophoresis of P1 (upper panel) and P2 (middle panel) fractions followed by Western immunoblotting with pCREB Ab Upstate. The P1 blot then subjected to stripping procedure, developed with secondary antibody to verify complete removal of the pCREB Ab, and reprobed with the CREB Ab CST (bottom panel). Most importantly, the positions of nuclear double bands detected with CREB Ab CST and pCREB Ab Upstate did not correspond to each other. Rather, the band detected with CREB Ab CST corresponded to the band detected with pCREB Ab Upstate. 2-D electrophoresis resolved the nuclear and mitochondrial pCREB IR's into two spots with such a big difference in apparent isoelectric points, that the y must be completely different proteins. The mitochondrial pCREB IR in fact represents another mitochondrial protein, which cross- reacts with the pCREB Ab's. lower upper 6) pCREB Ab’s stained mitochondria, in addition to nuclei, of glial cells in primary cortical cultures The primary neurona l cultures were made of new- born rat cortices, cytosine arabinoside added at day 2; and cultures used for imunofluorescence staining at day 3-6. Most of the cells were neurones, while glial elements accounted for less than 5 %. Antibodies against pCREB/ CREB, cytochrome oxidase ( COX) or astrocytic marker G FAP were used for staining, and the preparations were examined by confocal microscopy. pCREB antibody Upstate stained nuclei of all cells, but also mitochondria of glial elements, where it co-localized with COX. The mitochondrial signal w as evident even in unstimulated cultures (A), but was stronger when the cultures were challenged with forskolin (activator of protein kinase A) prior to fixation (B,C). If CREB Ab was used for staining, only faint nuclear signal could be obtained in this experimental system. 7) Three proteins precipitated by pCREB Ab from P2 fraction were identified as PDH Successful immunoprecipitation of the 'false' pCREB IR from P2 was consistently associated with 3 Coomassie-detectable protein bands of apparent MW 65.5, 42 and 31.5 kDa. These 3 bands were conclusively identified by MALDI-TOF MS as the E2, E1 alpha subunit, and E1 beta subunit components, respectively, of the well-known mitochondrial enzyme complex pyruvate dehydrogenase (PDH) In experiment A) we achieved identification of the 65.5 kDa band (MALDI-TOF MS spectrum shown). B) is another experiment where immunoprecipitation procedure was improved and the three bands were sent to Alphalyse A/S, (Odense, Denmar k, http://www.pick-n-post.com), which offers the MALDI-TOF MS protein identification as a commercial service; on the right is summary of results provided by the Alphalyse. 8) pCREB Ab reacts with purified pyruvate dehydrogenase Pyruvate dehydrogenase (PDH) purified from porcine heart (Sigma P-7032), subjected to Western immunoblotting with pCREB Ab Upstate: a single band of MW about 42 kDa (corresponds to the PDH E1 alpha subunit) is seen. T he rat PDH E1 alpha subunit sequence around phospho-Ser300 is similar to CREB sequence around phospho-Ser133. Bottom panel shows purified porcine PDH Sigma or rat brain P1 fraction subjected to Western blotting with pCREB Ab Upsta te that had been pre-incubated with excess PDH- or CREB-derived peptides as indicated. The peptide corresponding to phospho-Ser300 PDH completely blocked the pCREB Ab Upstate reactivity with purified PDH as well as 'false' pCREB IR in P1 fraction, while the same non-phosphorylated peptide was ineffective. 9) The cross-reacting epitope is phospho-Ser300 on PDH E1 alpha Conclusions We have found a pCREB-like immunoreactivity in brain mitochondria, which, however, after careful scrutiny turned out to be pyruvate dehydrogenase, rather than authentic CREB. This finding can explain most of the experimental evidence for mitochondrial localization of CREB, published previously; taken this into account there does not seem to be enough evidence for mitochondrial CREB localization. The commercial antibodies directed against CREB phospho-Ser133 cross-react with phospho-Ser300 on E1 alpha subunit of pyruvate dehydrogenase. The antibodies should be used with caution and some of the previous data based on their usage may need to be revisited. This work was published in Journal of Neurochemistry, Vol. 95, 2005, pp. 1446-1460 J.P. was Fellow of the Japan Society for the Promotion of Science while doing this work in Japan, and Fellow of the International Brain Research Foundation(IBRO) while continuing this study in Warsaw. In Cze ch Repu blic supported by a grant from the Grant A gency of the Czech Republic No. 303/02/1282, and grantsfrom th e Ministry of Education, Youth and Sports of the Czech Republic No. MSM 1111 0000 1, and No. MSM 0021620806. The (PDH) consists of three m ajor components: pyruv ate dehydrogenase/decarboxylase(E1) (EC 1.2.4.1.), dihydrolipoamide acetyltransferas e (E2) (EC 2.3.1.12.) lipoamide dehydrogenase(E3) (EC 1.6.4.3.), with stoichiometry 2 0-30 alpha2-beta2 tetramers + 6 homodime rs of E3 + 60 copies of E2 (Swissprotentry P08461). pyruvate dehydrogenase (Figure from Lodish et al. Molecular Cell Biology, 5th ed., W.H.Freeman Co., N.Y. 2004)

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Page 1: Apparent presence of Ser133-phosphorylated cyclic AMP res · Apparent presence of Ser133-phosphorylated cyclic AMP res ponse element binding protein (pCREB) ... in response to elevation

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od

ves

selfra

gm

en

tsin

the

P1,b

ut

only

very

few

nu

cle

iin

the

P2.

Lik

ew

ise,p

rese

nce

of

his

ton

eH

3d

ete

cte

dw

ithW

este

rnim

mu

no

blo

tting

was

res

tricte

dto

the

P1

fractio

n.S

eve

ral

dilu

tion

so

fP

1fra

ctio

nw

ere

inclu

ded

tosh

ow

that

the

fain

th

isto

ne

ban

din

P2

actu

ally

did

no

tre

pre

se

nt

mo

reth

an

ab

ou

t1%

of

his

ton

eco

nce

ntra

tion

inP

1.

5)

Ele

ctro

ph

ore

ticm

ob

ilityo

fm

itoc

ho

nd

rial

pC

RE

BIR

ma

rked

lyd

iffere

dfro

mn

uc

lea

rp

CR

EB

/CR

EB

IR

A):

The

CR

EB

IRo

nW

este

rnblo

tsde

tecte

dw

ithth

eC

RE

BA

bC

ST

inth

enuc

lear

fractio

nconsiste

ntly

app

eare

da

sa

double

ban

d,

likely

rep

resentin

gtw

oC

RE

Bsp

licin

gvaria

nts,

alp

ha

(the

upp

er

ban

d)

and

delta

(the

low

er

band

).Allo

the

rfra

ctions

app

are

ntly

con

tain

ed

only

delta

CR

EB

.T

heblo

tsdete

cte

dw

ithpC

RE

BA

bU

psta

teals

odis

pla

yed

ano

n-consta

ntdou

ble

band

inth

en

ucle

ar

P1

fractio

n,w

hile

inth

em

itochond

rialP

2fra

ctio

nth

ere

wa

salw

ays

asin

gle

band

with

positio

ncorre

spondin

gto

the

P1

low

erban

d.

B)

2-D

ele

ctroph

ore

sis

ofP

1(u

pper

pa

nel)

and

P2

(mid

dle

pan

el)

fractio

ns

follo

wed

by

We

stern

imm

uno

blo

tting

with

pC

RE

BA

bU

psta

te.

The

P1

blo

tth

en

subje

cted

tostrip

pin

gpro

cedure

,develo

pe

dw

ithsecondary

antib

od

yto

verify

com

ple

tere

mova

lof

the

pC

RE

BA

b,an

dre

pro

bed

with

the

CR

EB

Ab

CS

T(b

otto

mpan

el).

Mo

st

imp

orta

ntly,

the

po

sitio

ns

of

nu

cle

ar

do

ub

leb

an

ds

dete

cte

dw

ithC

RE

BA

bC

ST

an

dp

CR

EB

Ab

Up

sta

ted

idn

ot

co

rresp

on

dto

eac

ho

the

r.R

ath

er,

the

ban

dd

ete

cte

dw

ithC

RE

BA

bC

ST

co

rresp

on

ded

toth

eb

an

dd

ete

cte

dw

ithp

CR

EB

Ab

Up

sta

te.

2-D

ele

ctro

ph

ore

sis

reso

lve

dth

en

uclea

ran

dm

itoch

on

dria

lp

CR

EB

IR's

into

two

sp

ots

with

su

ch

ab

igd

iffere

nce

in

ap

pare

nt

isoe

lec

tricp

oin

ts,

that

the

ym

ust

be

co

mp

lete

lyd

iffere

nt

pro

tein

s.T

he

mito

cho

nd

rial

pC

RE

BIR

infa

ct

rep

resen

tsan

oth

er

mito

ch

ond

rial

pro

tein

,w

hic

hc

ross-

rea

cts

with

the

pC

RE

BA

b's

.

low

er

up

per

6)

pC

RE

BA

b’s

sta

ine

dm

itoch

on

dria

,in

ad

ditio

nto

nu

cle

i,o

fg

lialc

ells

inp

rima

ryc

ortic

alc

ultu

res

The

prim

ary

neu

rona

lcultu

res

were

made

ofnew

-born

ratcortice

s,cyto

sine

ara

bin

osid

ea

dded

atd

ay

2;a

nd

cultu

res

used

for

imuno

fluores

cence

stain

ing

at

day

3-6

.M

ostof

the

cells

were

neu

rone

s,w

hile

glia

le

lem

ents

acc

oun

ted

forle

ss

than

5%

.A

ntib

odie

sag

ain

stpC

RE

B/C

RE

B,

cyto

chro

me

oxid

ase

(CO

X)

or

astro

cytic

marke

rG

FA

Pw

ere

used

for

stainin

g,a

nd

the

pre

para

tions

were

exa

min

ed

by

confo

calm

icrosc

opy.

pC

RE

Bantib

od

yU

psta

testa

ined

nu

clei

ofa

llcells,

buta

lso

mito

chondria

ofglia

lelem

ents,

where

itco

-loca

lized

with

CO

X.T

he

mito

cho

ndria

lsig

nalw

as

evid

en

teve

nin

unstim

ula

ted

cultu

res

(A),

bu

tw

as

stron

ger

wh

en

the

cultu

res

were

challe

nge

dw

ithfo

rsko

lin(a

ctivato

ro

fpro

tein

kin

ase

A)

prior

tofix

atio

n(B

,C).

IfC

RE

BA

bw

as

used

for

stain

ing

,only

fain

tnu

clear

sign

alc

ould

be

ob

taine

din

this

experim

enta

lsy

stem

.

7)

Th

ree

pro

tein

sp

rec

ipita

ted

by

pC

RE

BA

bfro

mP

2fra

ctio

nw

ere

iden

tified

as

PD

H

Su

cc

es

sfu

lim

mu

no

pre

cip

itatio

no

fth

e'fa

lse'p

CR

EB

IRfr

om

P2

was

co

nsis

ten

tlya

sso

cia

ted

with

3C

oo

ma

ssie

-de

tec

tab

lep

rote

inb

an

ds

of

ap

pare

nt

MW

65

.5,

42

an

d3

1.5

kD

a.

Th

ese

3b

an

ds

we

rec

on

clu

sive

lyid

en

tified

by

MA

LD

I-TO

FM

Sas

the

E2

,E

1a

lpha

su

bu

nit,

an

dE

1b

eta

su

bu

nit

co

mp

on

en

ts,

res

pe

ctiv

ely

,o

fth

ew

ell-k

no

wn

mito

ch

on

dria

le

nzy

me

co

mp

lex

py

ruv

ate

deh

ydro

ge

na

se

(PD

H)

Ine

xperim

ent

A)

we

achie

ved

identific

atio

nofth

e65.5

kDa

band

(MA

LD

I-TO

FM

Ssp

ectru

msh

ow

n).

B)

isan

oth

er

exp

erim

entw

here

imm

unop

recip

itatio

npro

cedure

was

impro

ved

an

dth

eth

ree

bands

were

sentto

Alp

haly

seA

/S,(O

dense,D

enm

ark,

http

://ww

w.p

ick-n

-pos

t.com

),w

hich

offe

rsth

eM

ALD

I-TO

FM

Spro

tein

ide

ntifica

tion

as

acom

mercia

lservice

;on

the

right

issu

mm

ary

ofre

sults

pro

vide

db

yth

eA

lph

alys

e.

8)

pC

RE

BA

bre

ac

tsw

ithp

urifie

dp

yru

vate

deh

yd

rog

en

as

e

Pyru

va

tede

hyd

rogen

ase

(PD

H)

pu

rified

from

po

rcin

eh

eart

(Sig

ma

P-7

03

2),

su

bje

cte

dto

Weste

rnim

mu

no

blo

tting

with

pC

RE

BA

bU

ps

tate

:a

sin

gle

ban

do

fM

Wab

ou

t42

kD

a(c

orre

sp

on

ds

toth

eP

DH

E1

alp

ha

su

bu

nit)

issee

n.

Th

era

tP

DH

E1

alp

ha

subu

nit

sequen

cearo

und

pho

sph

o-S

er300

issim

ilar

toC

RE

Bsequ

ence

aro

und

phospho-S

er1

33

.

Bo

ttom

pane

lshow

spurifie

dpo

rcine

PD

HS

igm

aor

rat

bra

inP

1fra

ction

subje

cte

dto

Weste

rnb

lotting

with

pC

RE

BA

bU

psta

teth

ath

ad

be

en

pre

-incu

bate

dw

ithe

xcess

PD

H-

or

CR

EB

-deriv

ed

pep

tides

as

ind

icate

d.

Th

epep

tide

corre

spondin

gto

pho

spho

-Ser3

00

PD

Hcom

ple

telyblo

cked

the

pC

RE

BA

bU

psta

tere

activ

ityw

ithpurifie

dP

DH

as

well

as

'false

'pC

RE

BIR

inP

1fra

ctio

n,

while

the

sam

enon

-pho

sph

ory

late

dpe

ptid

ew

as

ineffe

ctive.

9)

Th

ecro

ss

-reactin

ge

pito

pe

isp

ho

sp

ho

-Ser3

00

on

PD

HE

1alp

ha

Co

nc

lus

ion

sW

eha

vefo

un

da

pCR

EB

-like

imm

un

ore

activity

inbra

inm

itocho

nd

ria,w

hic

h,

ho

we

ver,

afte

rcare

fuls

crutin

ytu

rned

outto

be

pyru

vate

de

hydro

gen

ase,

rath

er

tha

nauth

entic

CR

EB

.

Th

isfin

din

gca

nexp

lain

most

of

the

exp

erim

en

tale

vide

nce

for

mito

chon

dria

lloca

lizatio

no

fC

RE

B,pu

blish

ed

pre

viously

;ta

ke

nth

isin

toa

ccou

nt

there

do

es

no

tse

em

tob

een

ough

evid

enc

efo

rm

itoch

on

dria

lCR

EB

loca

lizatio

n.

Th

ec

om

me

rcia

lantib

odie

sdire

cte

dagain

st

CR

EB

pho

sp

ho-S

er1

33

cross-re

act

with

phospho

-Ser3

00

on

E1

alp

ha

sub

un

ito

fpyru

vate

de

hydro

gen

ase.

The

antib

odie

ssho

uld

be

use

dw

ithcau

tion

an

ds

om

eo

fth

epre

vio

us

data

bas

ed

on

the

irusag

em

ay

ne

ed

tob

ere

vis

ited.

Th

isw

ork

wa

sp

ub

lish

ed

inJo

urn

alo

fN

eu

roch

em

istry,

Vo

l.95

,200

5,

pp

.1

446

-1460

J.P.

was

Fello

wof

theJa

pan

So

ciety

forth

eP

rom

otion

ofS

cience

wh

iledo

ing

this

work

inJa

pan

,a

ndF

ellow

of

theIn

tern

ation

alBrain

Res

ea

rch

Fou

nda

tion

(IBR

O)

wh

ileco

ntinu

ingth

iss

tud

yin

Warsa

w.

InC

zech

Rep

ub

lics

upp

orte

dby

agra

ntfro

mth

eG

rantA

gen

cy

of

the

Cze

ch

Re

pub

licN

o.

303

/02/1

282

,an

dgra

nts

from

the

Min

istryof

Edu

ca

tion

,Yo

uth

an

dS

ports

ofth

e

Cze

ch

Re

pub

licN

o.M

SM

1111

000

01

,an

dN

o.M

SM

00

216

208

06.

The

(PD

H)

co

nsis

tso

fth

ree

ma

jorco

mp

one

nts:

pyru

vate

dehy

drog

enas

e/de

carb

oxyla

se

(E1)

(EC

1.2

.4.1

.),d

ihydro

lipo

am

ide

ac

etyltra

nsfe

ras

e(E

2)

(EC

2.3

.1.1

2.)

lipoa

mide

deh

ydro

gena

se

(E3

)(E

C1

.6.4

.3.),

with

stoich

iom

etry

20-3

0alp

ha

2-be

ta2

tetra

mers

+6

hom

odim

ers

of

E3

+60

cop

ies

ofE

2(S

wis

spro

ten

tryP

084

61

).

pyruv

ate

de

hyd

roge

nas

e

(Fig

ure

from

Lod

ish

et

al.

Mo

lecu

lar

Cell

Biolo

gy,

5th

ed

.,W

.H.F

ree

man

Co.,N

.Y.

20

04

)